Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

The isolation and characterisation of gas-cylinder membranes and α-granules fromAnabaena flos-aquae D 124

Summary Gas-cylinder membranes and -granules were isolated from cells of the blue-green alga,Anabaena flos-aquae D 124. Gas-cylinder membranes, which are 1,100 Å wide after isolation, show striations having a periodicity of 50 Å. The membranes appear globular in section with a spacing of about 40 Å between globules. There are strong indications that the globules are proteinaceous with molecular weights of 22,000±2,000. Possible homologies of these membranes with viral coat protein are discussed. -granules were shown by a variety of techniques to be polysaccharide.     

Fine structure of the vegetative thallus of the lichen,Peltigera polydactyla

Summary An investigation was made of the vegetative thallus of the lichen Peltigera polydactyla. Using a modified embedding technique it was shown that the ultrastructure of the mycobiont was not radically different from that of nonlichenized Discomycetes, and that the ultrastructure of the phycobiont was like that of the blue-green alga Nostoc. In addition to what were considered healthy blue-green algal cells other cells were present which obviously were breaking down. Well defined heterocysts were also present. No haustoria were found in the thallus.     

Growth and gas-vacuole development in vegetative cells of Anabaena flos-aquae

Summary Gas-vacuoles consisting of collections of gas-cylinders, and continuity of the plasma membrane with lamellae, are observed at all stages of growth of Anabaena flos-aquae. In day+4 cells, increased numbers of invaginations of the plasma membrane are observed and the lamellae tend to lie parallel to the cell wall. Some evidence is presented which suggests an associated synthesis of -granules and gas-cylinders by lamellae. Features of older cells are increased numbers of gascylinders, -granules, structured granules, and large intralamellar vesicles which appear to correlate with the presence of pinkish vacuoles as observed with the light microscope. The mean percentage of the volume of cells occupied by gas-vacuoles is about 20% during exponential growth when the doubling time is 56.5 hours, increasing to 34% in day+24 cells when growth is stationary.     

Environmental effects on the structure of the blue-green alga,Chlorogloea fritschii

Summary A study was made on the morphology and ultrastructure of Chlorogloea fritschii at different growth stages and under various environmental conditions. The environmental effects tested were a comparison of various light intensities, and the use of media, one with, and one without, a source of combined nitrogen. Both filamentous and endospore stages have the general ultrastructural features typical of a blue-green alga.The most marked differences found were in the arrangement of lamellae. These run parallel to the cell wall in young filaments, particularly in those grown at low light intensities. At the endospore stage these are much more scattered in arrangement.Two striking variations in this pattern were found. One cell type, possibly a spore, showed, along with other interesting features, marked reticulation of the lamellae. Material subcultured in the dark with sucrose for 3 years had the lamellae scattered throughout the cell.Differences were found also in the abundance of -granules (probable polyglucoside bodies). These differences could to a certain extent be correlated with the growth conditions used.A brief comparison of Chlorogloea with other blue-green algae is included. Based on this, tentative hypotheses are presented concerning correlations between ultrastructural features and taxonomic groupings.     

Comparative structure of the gas-vacuoles of blue-green algae

Summary An investigation was made of 5 species of blue-green algae reported to contain gas-vacuoles. All organisms were grown and harvested under standard conditions. Gas-vacuoles were characterised as reddish structures which are destroyed by applying pressure. Using a simple direct preparation technique gascylinders were observed with the transmission electron microscope in gas-vacuolate cells. Gas-vacuoles were present in the strains of Anabaena flos-aquae, Gloeotrichia echinulata and Oscillatoria agardhii studied and absent from Microcystis aeruginosa and Nostoc linckia. The reddish, refractile central area of N. linckia and M. aeruginosa cells was tentatively identified as nucleoplasm. Gas-vacuoles are collections of gas-cylinders 70 m wide, which in A. flos-aquae and G. echinulata are clearly bounded by photosynthetic lamellae and associated with -granules. The presence of bounding photosynthetic lamellae in these species is suggested as a causal factor of the unusual optical properties of their gas-vacuoles. The range of lengths of gas-cylinders in G. echinulata and O. agardhii is from 100 m to 500 m and in A. flos-aquae it is from 100 m to 1300 m. The percentage of cell volume occupied by gas-vacuoles was estimated by direct measurement. In A. flos-aquae and G. echinulata it was 22%. In O. agardhii gas-cylinders were not clearly associated with photosynthetic lamellae and -granules and occupied 39% of cell volume. Gascylinder membranes showed reasonable preservation in KMnO₄ and excellent preservation in OsO₄. The widths of membranes after treatment with these two fixatives was 3 m and 2 m respectively.     

Characterization of crystals of the thermostable DNA polymerase I from thermus aquaticus

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 Å resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermo stability in proteins. © 1995 Wiley-Liss, Inc.     

Determination of the Structure of the Catabolic N-Succinylornithine Transaminase (AstC) from Escherichia coli

Escherichia coli possesses two acyl ornithine aminotransferases, one catabolic (AstC) and the other anabolic (ArgD), that participate in L-arginine metabolism. Although only 58% identical, the enzymes have been shown to be functionally interchangeable. Here we have purified AstC and have obtained X-ray crystal structures of apo and holo-AstC and of the enzyme complexed with its physiological substrate, succinylornithine. We compare the structures obtained in this study with those of ArgD from Salmonella typhimurium obtained elsewhere, finding several notable differences. Docking studies were used to explore the docking modes of several substrates (ornithine, succinylornithine and acetylornithine) and the co-substrate glutamate/α-ketogluterate. The docking studies support our observations that AstC has a strong preference for acylated ornithine species over ornithine itself, and suggest that the increase in specificity associated with acylation is caused by steric and desolvation effects rather than specific interactions between the substrate and enzyme.     

Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg^2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K_D ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC₅₀ ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N ⁹ substitution, or tautomerization arising from N ⁷ substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N ⁹-H···Val46 hydrogen bond, combined with the limited space available around the N ⁹ position. The water-filled pocket under the N ⁷ position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N ⁷ (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K_D ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N ⁷ position towards the Mg²⁺-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg²⁺ centres or Mg²⁺ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.